Toxicity and biological effects of three Egyptian isolates of baculovirus on the cotton leaf worm , Spodoptera littoralis ( boisd )

The toxicity of three different isolates of Egyptian baculovirus namely NPVGiza, NPVCairo and NPVAlex to Spodoptera littoralis larvae and their effects on some biological aspects of adults were studied. The second and fourth larval instars were highly susceptible to NPVCairo than to the other isolates. The second larval instar was highly susceptible than the fourth larval instar. NPVCairo induced larval, pupal and adult malformations. It was found that the three NPV's decreased the moth longevity, fecundity, egg hatchability and altered the sex ratio.


INTRODUCTION
Human attempts at insect control have changed over time from natural to synthetic chemical control, and now again we look to natural control methods.Longterm exposure to synthetic insecticides causes many healthy problems, such as cancer, immunotoxicity and other complicated problems to man and animals.Insecticides are not specific in their action; they kill non-target and the beneficial insects, so that biological control has received more attention.
There are many biological control agents, such as bacteria, fungi and viruses (Jones, 1990 andPawar, et al., 1991).Baculoviridae includes nuclearpolyhedrovirus (NPV) which has polyhedron-shaped occlusion bodies.The baculovirus isolates have a limited host range, and infect only closely related species as for insects mostly of order Lepidoptera.
The main objective of the present study was to search for natural nucleopolyhedrovirus isolate(s) with better insecticidal characteristics.To accomplish this objective, the following strategy was followed: dead or diseased lepidopterous larvae were collected and viral isolation, purification and propagation were then carried out.After purification, the different viral isolates were tested for their insecticidal activities and to study their effect on malformations and some biological aspects of Spodoptera littolaris.

Colony of Spodoptera littoralis
A laboratory colony of the Egyptian cotton leafworm, S. littoralis was obtained from the Department of cotton leafworm, Institute of Plant Protection, Agricultural Research Center, Ministry of Agriculture, Dokki, Giza, Egypt.Larvae were fed semisynthetic diet according to Shory and Hale (1965).The colony was kept at 25 ± 2 °C, 65-70 % RH. under highly controlled conditions to avoid contamination.Such colony was used for virus propagation, bioassay test and biological observation.

Virus isolation, multiplication and purification
During 2005-2006 larvae of S. littoralis were collected manually from cotton and maize fields in three Governorates (Giza, Cairo and Alexandria).Larvae were examined for virus presence and the diseased ones (displaying symptoms of baculovirus infection) were immediately frozen for viral isolation.
The diseased larvae were allowed to liquefy for several days at 25 ± 2°C in plastic beakers covered with Parafilm.A crude extract was prepared by adding distilled water to each beaker and filtering the resulting mixture through a layer of naylon fabrics to remove tissue debris.The crude extract was stored at-80 °C until further processing as described by Fuxa, et al., (1999).
The three isolates of SpliNPV were propagated by feeding third-instar larvae on semisynthetic diet, which was contaminated with virus occlusion bodies (OBs).OBs were harvested and purified from larvae cadavers as described by El Salamouny (1998).

Bioassay test
Suspension of the highly purified NPV was tested against early second and fourth larval instars.The semisynthetic diet without formalin was poured before cooling into 3 g plastic cups forming a layer of 0.5 cm.thick, which on cooling gave very smooth surface.A standard volume of 250µl of virus suspension was pipetted on to the diet surface, distributed and then left to the air.In the control treatment virus suspension was replaced by distilled water.Five different concentrations ranging from 1.8x10 7 to 1.8x10 3 polyhedra / ml were tested.Three replicates each of 10 larvae were used for each virus concentration.All treatments were incubated at 25°C, 60-70% RH. and natural photoperiod.Mortality was recorded at the tenth day post-infection.
Mortality data were corrected using the Abbott's formula (1925) and Lethal concentrations (LC 25 and LC 50 ) in Polyhedral inclusion body (PIB) / ml were calculated according to the method of Litchfield and Wilcoxon (1949).

Biological observations
Early second larval instars treated with the prdetermined LC 25 of the three NPV isolates were divided into two groups and observed till adult emergence.In the first group, longevity, and fecundity of the resultant adult females and egg hatchability were recorded.Three replicates each of 10 larvae were used for each virus.In the 2 nd group, malformations and morphological abnormalities in larval, pupal and adult stages were observed.

Statistical analysis
Data were presented as mean± standard error (S.E.).The mean values were compared by t-test using the "Graphpad quick calculation" computer program (http://www.Graphpad.com).A P-value of ≤ 0.05 was considered indicative of statistical significance.

Susceptibility of Spodoptera littoralis larvae to the three NPV isolates
Results obtained from bioassay tests (Table 1) demonstrated that the 2 nd larval instar was more susceptible to the three tested viral isolates (NPV Giza , NPV Cairo and NPV Alex ) than the 4 th larval instar.Further more, both 2 nd and 4 th larval instars exhibited higher susceptibility to NPV Cairo than to the other isolates.

Toxicity and biological effects of baculovirus on the cotton leaf worm 221
Different susceptibility levels of S. littoralis larvae to NPV's were observed by several authors (Klein and podoler, 1978;Komolpith and Ramakrishnan, 1975;Pawar and Ramakrishnan, 1975;Seufi andOsman 2005 andSeufi 2008).Such difference in susceptibility may be due to the difference in larval age, the number of virions contained in occlusion bodies, the method of virus administration, the feeding habit of the insect, the difference in viral strain and /or the lower number of propagation cycles of different isolates (Payne, 1982;Seufi, 2002).The results were agreeable to that of Stairs (1965), who found that the susceptibility decreased markedly as larvae of Malacosoma disstria grew older.In parallel, Duan and Otvos (2001) reported that mortality was higher when younger larvae of Choristonura fumiferana were used.

Effect of virus isolates on the biological aspects of Spodoptera littoralis adult female
The treatment of the 2 nd larval instar with LC 25 of the three NPV's (Table 2) decreased the moth longevity, fecundity and egg hatchability.In addition, the three NPV isolates altered sex ratio in favor of males (♂♂: total = 0.94, 0.94 and 0.77 for Giza, Cairo and Alexanria isolates, respectively) in comparison to control (♂♂: total = 0.17) Based on the hypothesis that the efficacy of the viral biopesticide is judged by observing percent mortality and abnormalities at different applied doses (Prasad and Wadhwani, 2006).NPV Cairo was more potent than other isolates, as the adult moth died after emergence.In parallel, Rothman and Myers (1996) reported that viral diseases of lepidoptera are characterized by their ability to kill infected host and to reduce the fitness of individuals that survive infection.
In consistence with our results, Duan and Otvos (2001) noticed that sublethal doses of Choristonura fumiferana NPV decreased longevity of adults and reduced proportions of females among survivors.However, Young (1990) reported that NPV had little effect on pupal mortality, sex ratio and hatchability when applied to 4 th larval instars of Spodoptera ornithogalli.

Malformation and morphological abnormalities
Two NPV isolates (NPV Cairo and NPV Alex ) were observed for induction of malformed S. littoralis.Generally, the present study revealed that the resultant abnormal larvae (Figs. 1 b-f) and pupae (Figs. 2 b-f) failed to transform to the next developing stages during the normal period as in the case of control individuals.Several malformations or abnormalities were observed in treated larvae and represented by: 1-larval-pupal intermediate with larval head, thoracic legs and pupal abdomen attached with larval exuvium, (Fig. 1-b ).2-dwarfed larval pupal intermediate with larval head, thoracic legs and balloon shaped pupa from posterior end, (Fig. 1-c).3-pupa attached with larval exuvium from posterior end, (Fig. 1-d).4-larval-pupal intermediate with clear appearance of larvae, (Fig. 1-e).5-dwarfed larval pupal intermediate with larval head, thoracic legs and pupal abdomen, (Fig. 1-f).Retardation in larval development was noticed in some 2 nd larvae treated with NPV Alex as they remained in the same instar until dying (Fig. 1 g).6-malformed pupae that failed to transform to normal adult were represented in (Fig. 2 b-f ).7-Figure (3-b) show malformed adults with crumbled wings as compared to the normal adult (Fig. 3-a).

Table ( 1
): Susceptibility of Spodoptera littoralis larvae to three nucleopolyhedrovirus isolated from Giza, Cairo and Alex.

Table ( 2
): Effect of LC 25 of NPV Giza , NPV Cairo and NPV Alex .isolates on the biological aspects of Spodoptera littoralis adult female under laboratory conditions.In each row, means indicated by (*) are significantly different as compared to the control and those with different letters are significantly different from each other ( t-test, P < 0.05).